Enzymatic Assemblies Disrupt Membrane and Target Endoplasmic

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1 Supporting Information for Enzymatic Assemblies Disrupt Membrane and Target Endoplasmic Reticulum (ER) for Selective Cancer Cell Death Zhaoqianqi Feng, Huaimin Wang, Shiyu Wang, Qiang Zhang, Xixiang Zhang, Avital A. Rodal and Bing Xu *, Department of Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States. Department of Biology, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States. Physical Science and Engineering Division (PSE), King Abdullah University of Science and Technology (KAUST), Thuwal , Saudi Arabia Content S1. Experimental materials and instruments... 4 S2. Synthesis and characterization of the precursors... 5 Scheme S1. Molecular structure of precursors 1P, 2P, 3P, 4P, F1P, F4P and their corresponding dephosphorylation products 1, 2, 3, 4, F1, F4. Scheme S2. Synthesis route of 1P. S3. TEM sample preparation S4. Static light scattering measurement... 8 S5. CMC measurements S6. Cell culture and MTT assay... 8 S1

2 S7. Liposome co-sedimentation... 9 S8. LDH assay S9. Time-dependent Western blot S10. PathScan apoptosis multi-target sandwich ELISA S11. Description of videos Fig. S1. 1 H NMR spectrum of 1P in DMSO-d6 Fig. S2. 31 P NMR spectrum of 1P in DMSO-d6 Fig. S3. 1 H NMR spectrum of 2P in DMSO-d6 Fig. S4. 31 P NMR spectrum of 2P in DMSO-d6 Fig. S5. 1 H NMR spectrum of 3P in DMSO-d6 Fig. S6. 31 P NMR spectrum of 3P in DMSO-d6 Fig. S7. 1 H NMR spectrum of F1P in DMSO-d6 Fig. S8. 31 P NMR spectrum of F1P in DMSO-d6 Fig. S9. 1 H NMR spectrum of F4P in DMSO-d6 Fig. S P NMR spectrum of F4P in DMSO-d6 Fig. S11. Optical images of 1P at the concentration of 0.5 wt% before and after treating with ALP (1 U/mL). S2

3 Fig. S12. LC-MS spectra of 1P (0.5 wt%) after treating with 1 U/mL ALP. Fig. S13. CMCs for 1P without or with the treatment of ALP (1 U/mL). Fig. S14. TEM images of the nanostructures formed by 1P at different concentrations, after adding ALP (1 U/mL) (scale bar 50 nm). Fig. S15. Two independent experiments of cell viability of HeLa, A2780cis, OVSAHO, or HS-5 cells treated with 1P. Fig. S16. Cell viability of HeLa cells treated with 1P (200 µm) in the presence of phosphatase inhibitors. ([ALP] = 3 U/mL, [TNAP inhibitor(dqb)] = 2 μm, [Phe] = 3 mm. Fig. S17. The liposome binding capability of 1P (25 µm), with or with-out the treatment of ALP. Data obtained by triplicate measurements (n = 3) and presented as mean ± SEM. Fig. S18. TEM images of 2P or 3P at the concentration of 0.5 wt%. Fig. S19. The liposome binding capability of 1P, 2P or 3P (25 µm) after the treatment of ALP. Data obtained by triplicate measurements (n = 3) and presented as mean ± SEM. Fig. S20. TEM images of F1P (200 µm) before and after treating with 1 U/mL ALP. Fig. S21. CLSM images of HeLa cells treated with F4P (200 µm) for 1h after staining with Hoechst (nuclei, blue). Scale bar: 10 μm. Fig. S22. CLSM images of HS-5 cells treated with F1P (200 µm) for 1 h after staining with Hoechst (nuclei, blue). Scale bar: 10 μm Fig. S23. Time-dependent Western blot analysis of ER-stress markers and caspase 8 after treating HeLa cells with 1P (50 μm) at different time (i.e., 0, 3, 6, 12, 24 or 36 h). S3

4 Fig. S24. Cell viability of HeLa cells treated with 1P and with 1P plus N-acetylcysteine (1mM). Fig. S25. Time-dependent Western blot analysis of ER-stress markers and caspase 8 after treating A2780cis cells with 1P (100 μm) at different time (i.e., 0, 3, 6, 12, 24 or 36 h). Fig. S26. Time-dependent PathScan apoptosis multi-target sandwich ELISA of HeLa cells treated with 1P (100 μm) at different time. S1. Experimental materials and instruments 2-Cl-trityl chloride resin ( mmol/g), HBTU, Fmoc-OSu, and other Fmoc-amino acids were obtained from GL Biochem (Shanghai, China). Other chemical reagents and solvents were purchased from Fisher Scientific. Alkaline phosphatase was purchased from Biomatik (Cat. No. A1130, Alkaline Phosphatase [ALP], > 1300U/MG, in 50% Glycerol.), RPMI 1640 Medium from ATCC, Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and penicillin/streptomycin from Gibco by Life Technologies, and 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) from ACROS Organics. All precursors were purified with Water Delta600 HPLC system, equipped with an XTerra C18 RP column and an in-line diode array UV detector. The LC-MS spectrums were obtained on Waters S4

5 Acquity Ultra Performance LC with Waters MICROMASS detector, ultraviolet-visible (UV) spectra on JASCO J-810 spectrophotometer, and 1 H-NMR spectra on Varian Unity Inova 400. CLSM images were obtained using Zeiss LSM 880 confocal microscopy at the lens of 63 with oil. Live-cell videos were obtained with Smaug (a laser scanning confocal inverted Nikon Ti-E-PFS3 eclipse microscope with several solid-state lasers, a 7-LED Light Engine array, an scmos camera (Andor Zyla), a Nikon C2 point-scanning head, and a 60 oil objective (n.a. 1.4). The lasers used are 405 and 488 nm. S2. Synthesis and characterization of the precursors We synthesized Fmoc-Tyr(PO3H2)-OH/ Fmoc-Ser(PO3H2)-OH based on previous work for directly use of solid phase peptide synthesis (SPPS). Using SPPS, we synthesized 1P, 2P, 3P, F1P and F4P (Scheme S1) based on reported protocols. 1 Scheme S2 shows the synthetic route for 1P. The synthetic route of other compounds are the same with 1P. The compounds were purified by reverse phase HPLC using acetonitrile (0.1% TFA) and double-distilled water (0.1% TFA) as the eluents. S5

6 Scheme S1. Molecular structures of precursors 1P, 2P, 3P, 4P, F1P, F4P and their corresponding dephosphorylation products 1, 2, 3, 4, F1, F4. S6

7 Scheme S2. Synthesis route of 1P. LC-MS (ESI): 1P (m/z): C46H52N7O10P, calc ; observed [M-H] P (m/z): C45H50N7O10P, calc ; observed [M-H] P (m/z): C45H50N7O10P, calc ; observed [M-H] F1P (m/z): C43H50N11O13P, calc ; observed [M-H] F4P (m/z): C37H46N11O13P, calc ; observed [M-H] S3. TEM sample preparation After placing 5 µl samples on 400 mesh copper grids coated with continuous thick carbon film (~ 35 nm) which is glowed discharged, we washed the grid with ddh2o twice and stained the sample loaded grid with a large drop of the UA (uranyl acetate) and allow to dry in air. HRTEM and TEM S7

8 images were obtained with TITAN FEG scanning transmission electron microscope and Morgagni 268 transmission electron microscope, respectively. S4. Static light scattering measurement We performed static light scattering on ALV (Langen, Germany) goniometer and correlator system with a 22 mw HeNe (λ = 633 nm) laser and an avalanche photodiode detector. The samples of 1P at the concentration of µm, in ph 7.4 PBS buffer were prepared with or without the addition of 1 U/mL ALP for 24 hours. The light scattering degree is 30. S5. CMC measurements A series of precursor (1P, 2P, 3P) solutions from the concentration of 2 mm to 0.5 µm was prepared in ph 7.4 PBS buffer, with or without the treatment of 1 U/mL ALP. After incubating with Rhodamine 6G (5 µm), the λmax was determined by measuring the absorbance from 520 to 540 nm using a Biotek Synergy 4 hybrid multi-mode microplate reader. S6. Cell culture and MTT assay Cell culture: HeLa cells and HS-5 cells were purchased from American Type Culture Collection (ATCC, USA). OVSAHO cells and A2780cis cells were given by Dr. Daniela Dinulescu group. HeLa cells were cultured in Minimal Essential Medium (MEM) supplemented with 10% v fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/ml streptomycin; HS-5 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% v fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/ml streptomycin OVSAHO cells and A2780cis cells were S8

9 cultured in RPMI 1640 Medium supplemented with 10% v/v fetal bovine serum, 100 U/mL penicillin and 100 μg/ml streptomycin. All cells were maintained at 37 C in a humidified atmosphere of 5% CO2. MTT assay: We determined the cytotoxicity of precursors using MTT assay. Cells were seeded in 96-well plates at cells/well for 24 hours followed by culture medium removal and subsequently addition of culture medium containing different concentration of the precursors (immediately diluted from fresh prepared 10 mm stock solution). After 24/48/72 hours, 10 μl MTT solution (5 mg/ml) was added to each well and incubated at 37 C for another 4 h. Then 100 μl of SDS-HCl solution was added to stop the reduction reaction and dissolve the formazan. The absorbance of each well at 595 nm was measured by a DTX880 Multimode Detector. The results were calculated as cell viability percentage relative to untreated cells. Data were obtained by from three independent wells (n = 3). S7. Liposome co-sedimentation Lipid co-sedimentation assays were conducted as previously described. 2 Liposomes were swelled from dried lipid films, composed of 79% phosphatidylcholine (PC), 15% phosphatidylethanolamine (PE), 5% phosphatidylserine (PS) and 1% phosphatidylinositol 4,5-bisphosphate (PIP2) from Avanti Polar Lipids, in 20 mm Hepes ph 7.5 and 100 mm NaCl. Compounds were then mixed with 0.4 mg/ml liposomes or buffer alone, incubated for 1 h at room temperature, followed by pelleting for 20 min at 18,000 g at 4 C. Pellets and supernatants were separated and detected using LC-MS. No pelleting of compounds was observed in the absence of liposomes at the concentrations reported S9

10 (20 µm, 25 µm). S8. LDH assay LDH release (%) was measured using Pierce LDH Cytotoxicity Assay Kit purchased from Thermo Scientific, using the protocols as following: 1. Seed HeLa cells at cells/well in triplicate wells in a 96-well plate for 24 hours to allow attachment. 2. Remove culture medium and incubate the cells with the precursors (1P, 2P, 3P) at different concentrations. Add 10 μl Lysis Buffer (10X) 3. After incubation with precursors at different time, transfer 50 μl of each sample medium to a 96-well plate. 4. Add 50 μl Reaction Mixture to each sample well and mix gently. 5. After incubating the plate at room temperature for 30 minutes protected from light, add 50 μl of Stop Solution to each sample well and mix by gentle tapping. 6. Measure the absorbance at 490 and 680 nm using a Biotek Synergy 4 hybrid multi-mode microplate reader. Before calculation of LDH release, subtract the 680 nm absorbance value (background) from the 490 nm absorbance [(LDH at 490 nm) - (LDH at 680 nm)]. The LDH release (%) was calculated as percentage relative to maximum LDH activity controls. S9. Time-dependent Western blot 1. After the HeLa cells grows to 80 90% confluence in 10 cm culture dish, treated cells with 1P (50 µm) at different time point. S10

11 2. Remove the culture medium and wash the cells by pre-cold PBS for three times, then add 0.5 ml ice-cold cell lysis buffer plus protease inhibitors to each plate and incubate on ice for 5 min. 3. Scrape cells off the plate and transfer to a 1.5 ml tube. 4. Freeze-thaw 3 cycles of collected cell fraction. Centrifuge for 20 min (12,000 rpm) at 4 C and transfer the supernatant to a new tube. The supernatant is the cell lysate. 5. Perform the standard Western blot. S10. PathScan apoptosis multi-target sandwich ELISA The PathScan apoptosis multi-target sandwich ELISA kit was purchased from Cell Signaling Technology, Inc. (CST). Using the procedures which were described in S9 to collect the cell lysate, we followed the detail protocol as provided by cell signaling technology for EISA test. S11. Description of videos Video S1 Time-lapse of live HeLa cells after incubated with 1P (200 µm) after staining the plasma membranes with lipid raft probe (10 μm) for 1 h. Video S2 Time-lapse of live HeLa cells incubated with F1P (200 µm) after staining the nucleus with Hoechst S11

12 Figure S1. 1 H NMR spectrum of 1P in DMSO-d6 Figure S2. 31 P NMR spectrum of 1P in DMSO-d6 S12

13 Figure S3. 1 H NMR spectrum of 2P in DMSO-d6 Figure S4. 31 P NMR spectrum of 2P in DMSO-d6 S13

14 Figure S5. 1 H NMR spectrum of 3P in DMSO-d6 Figure S6. 31 P NMR spectrum of 3P in DMSO-d6 S14

15 Figure S7. 1 H NMR spectrum of F1P in DMSO-d6 Figure S8. 31 P NMR spectrum of F1P in DMSO-d S15

16 Fig. S9. 1 H NMR spectrum of F4P in DMSO-d6 Fig. S P NMR spectrum of F4P in DMSO-d6 S16

17 Fig. S11. Optical images of 1P at the concentration of 0.5 wt% before and after treating with ALP (1 U/mL). Fig. S12. LC-MS spectra of 1P (0.5 wt%) after treating with 1 U/mL ALP. Fig. S13. CMCs for 1P without or with the treatment of ALP (1 U/mL). S17

18 Fig. S14. TEM images of the nanostructures formed by 1P at different concentrations, after adding ALP (1 U/mL) (scale bar 50 nm). Fig. S15. Two independent experiments of cell viability of HeLa, A2780cis, OVSAHO, or HS-5 cells treated with 1P. S18

19 Fig. S16. Cell viability of HeLa cells treated with 1P (200 µm) in the presence of phosphatase inhibitors. ([ALP] = 3 U/mL, [TNAP inhibitor(dqb)] = 2 μm, [Phe] = 3 mm. Fig. S17. The liposome binding capability of 1P (25 µm), with or with-out the treatment of ALP. Data obtained by triplicate measurements (n = 3) and presented as mean ± SEM. Fig. S18. TEM images of 2P or 3P at the concentration of 0.5 wt%. S19

20 Fig. S19. The liposome binding capability of 1P, 2P or 3P (25 µm) after the treatment of ALP. Data obtained by triplicate measurements (n = 3) and presented as mean ± SEM. Fig. S20. TEM images of F1P (200 µm) before and after treating with 1 U/mL ALP. S20

21 Fig. S21. CLSM images of HeLa cells treated with F4P (200 µm) for 1 h after staining with Hoechst (nuclei, blue). Scale bar: 10 μm. Fig. S22. CLSM images of HS-5 cells treated with F1P (200 µm) for 1 h after staining with Hoechst (nuclei, blue). Scale bar: 10 μm S21

22 Fig. S23. Time-dependent Western blot analysis of ER-stress markers and caspase 8 after treating HeLa cells with 1P (50 μm) at different time (i.e., 0, 3, 6, 12, 24 or 36 h). Fig. S24. Cell viability of HeLa cells treated with 1P and with 1P plus N-acetylcysteine (1mM). Fig. S25. Time-dependent Western blot analysis of ER-stress markers and caspase 8 after treating A2780cis cells with 1P (100 μm) at different time (i.e., 0, 3, 6, 12, 24 or 36 h). S22

23 Fig. S26. Time-dependent PathScan apoptosis multi-target sandwich ELISA of HeLa cells treated with 1P (100 μm) at different time. Reference (1) Shi, J.; Du, X.; Yuan, D.; Zhou, J.; Zhou, N.; Huang, Y.; Xu, B. Biomacromolecules 2014, 15, 3559; Ottinger, E. A.; Shekels, L. L.; Bernlohr, D. A.; Barany, G. Biochemistry 1993, 32, (2) Becalska, A. N.; Kelley, C. F.; Berciu, C.; Stanishneva-Konovalova, T. B.; Fu, X. F.; Wang, S. Y.; Sokolova, O. S.; Nicastro, D.; Rodal, A. A. Mol. Biol. Cell 2013, 24, S23